staining methods

1. STAINING :IT IS A
BIOCHEMICAL TECHNIQUE OF ADDING SPECIFIC
DYE TO A SUBSTRATE (DNA,PROTEINS,LIPIDS)TO
QUALIFY OR QUANTIFY THE PRESENCE OF A
SPECIFIC COMPOUND
THE STAINS MOST COMMONLY USED FOR STAINING
OF BLOOD FILMS ARE ROMANOWSKY STAINS.

2. ROMANOWSKY STAIN :IT IS NAMED AFTER DMITRI
LEONOVICH ROMANOWSKY WHO INVENTED IT IN
1891
DEFINITION:A STAIN MADE FROM WATER SOLUBLE
EOSIN,METHYLENE BLUE AND ACETONE FREE
METHANOL .

3. VARIOUS STAINS FOR PERIPHERAL BLOOD FILM:
Romanowsky stains are universally employed for
staining of blood films. All Romanowsky
combinations have two essential ingredients i.e.
methylene blue and eosin .

4. Methylene blue:
1)Cationic dye
2) Basic dye
3) Blue-Purple colour
Structures that stain with methylene blue are termed
as Basophilic.

5. Eosin:
1) Anionic dye
2)Acid dye
3)Pink red-colour
Structures that stain with eosin are termed
Eosinophilic.

6. Methylene blue combines with anionic components of the cell .eg:DNA ,and stain
these blue.
Eosin combines with cationic components of the cell .
Eg:cytoplasm and stain them red .
Then there occurs stain –stain interaction.
This composition and mode of action allows romanowsky stains to reveal the subtle
differences in shades of staining.

7. A well stained –smear with any of romanowsky stains shows following features :
1)Red blood cells:Pink red (or)Deep red colour.
2)Polychromatic cells(reticulocytes):Gray blue colour.
3) Neutrophils :Pale,Pink cytoplasm,Purple Granules .
4)Eosinophils:Pale,Pink cytoplasm,orange red granules

8. 5) Basophils:Blue cytoplasm,Dark blue-Violet Granules .
6) Monocytes:Gray –Blue cytoplasm.
7)Lymphocytes:Dark blue cytoplasm.
8) Platelets:Purple Colour.

11. To preserve the morphology of the cells, films
must be fixed as soon as possible after they have
dried.
It is important to prevent contact with water
before fixation is complete.
Methyl alcohol (methanol) is the choice, although
ethyl alcohol ("absolute alcohol") can be used.

12. Methylated spirit (95% ethanol) must not be used as
it contains water.
To fix the films, place them in a covered staining jar or
tray containing the alcohol for 2-3 minutes.
In humid climates it might be necessary to replace the
methanol 2-3 times per day; the old portions can be
used for storing clean slides.

13. VARIOUS STAINS INCLUDED UNDER ROMANOWSKY
STAINS ARE AS FOLLOWS:
1)Leishman stain
2)Giemsa stain
3)Wright stain

14. 4)Field stain
5)Jenner stain
6)JSB stain

15. Among the above mentioned stains ,Jenner is the
simplest and Giemsa is the most complex.
Leishman stain it occupies intermediate position and it
is still widely used in the routine staining of blood
films.

16. LEISHMAN STAIN:
PRINCIPLE:
It is used in microscopy for staining blood smears.It
provides excellent stain quality .
COMPONENTS:It contains methylene blue and eosin
in methanol(acetone free)in the ratio of 1.5gm/1
litre.
It is generally used to differentiate and identify
leucocytes,malaria parasites etc.

17. LEISHMAN STAIN:
Preparation
Dissolve 0.2 g of powdered Leishman’s dye in 100 ml
of acetone-free methyl alcohol in a conical flask.
Warm it to 50°C for half an hour with occasional
shaking.
Procedure for staining
1)Pour Leishman’s stain dropwise (counting the drops)
on the slide and wait for 2 minutes. This allows
fixation of the PBF in methyl alcohol.

18. 2)Add double the quantity of buffered water dropwise
over the slide (i.e. double the number of drops).
3) Mix for 8 minutes
4)Wash in water for 1 to 2 minutes.
5)Dry in air and examine under oil immersion lens of
the microscope.

19. GIEMSA STAIN:
other
PRINCIPLE:
It is used in diagnosis of malaria and
parasites ,also used in cytogenetics.
Components for giemsa stain :
1)Methylene blue
2)Azure B
3)Sodium salt of Eosin Y.

20. GIEMSA STAIN
Preparation
Stock solution of Giemsa stain is prepared by mixing 0.15 g of Giemsa powder in
12.5 ml of glycerine and 12.5 ml of methyl alcohol.
Before use dissolve one volume of stock solution in nine volumes of buffered
water (dilution 1:9).

21. Procedure:
1) Fill staining dish with staining solution
2) Place thin blood film(useful for investigation of
anemia ,infections etc,) and thick blood films(useful
for investigation of malaria and microfilaria) into the
staining dish

22. 3) Stain blood slides for 45 minutes
4) Wash in water.
5)Dry it and examine under oil immersion lens of the
microscope.

23. WRIGHT'S STAIN:
PRINCIPLE:
Wright’s stain is used to
nuclear and/or
RBCs, WBCs,
morphology of
and
differentiate
cytoplasmic
platelets,
parasites.
Materials:
1)Wright stain powder-1.0gm.
2)methanol(must be water free)-400ml.

24. Preparation:
1)Weigh the wright powder and transfer it to a dry
brown bottle.
2)Add a few glass beads(to assit in dissolving the dye)
3)Using a dry cylinder ,measure the methanol and add
to this stain.

25. 4)Mix well at intervals until the powder is completely dissolved.
5)Warming the solution in a 37 c water bath will help the dye to dissolve.
6)label the bottle and mark it flammable and toxic.

26. Procedure:
Thin blood films (only) – Dip Method
1. Dip air dried blood film in undiluted stain for 15 to 30
seconds
(double the staining time for bone marrow smears).
2.Decolorize the stained smears by immersion in distilled
or deionized water and air dry.
3. Let air dry in a vertical position.

27. Thin Blood films (only)-Rack Method.
1)Lay air dried slides on staining rack and flood with
stain,stain for 10 to 15 seconds(double the staining time
for bone marrow smears).
2)Add an equal volume of deionized/distilled water and
stain for 10 seconds.
3)Rinse the slide by dipping in deionized/distilled water
for 30 seconds.

28. 4. Allow the film to air dry.
5.Dip air dried blood film in undiluted stain for 15 to 30
seconds (double the staining time for bone marrow
smears).
6.Decolorize the stained smears by immersion in distilled
or deionized water and air dry
7.Let air dry in a vertical position.

29. THICK BLOOD FILMS (ONLY):
Allow film to air dry thoroughly for several hours
or overnight. Do not dry films in an incubator
or by heat, because this will fix the blood and
interfere with the lysing of the RBCs.
Note: If a rapid diagnosis of malaria is needed,
thick films can be made slightly thinner than
usual, allowed to dry for 1 h, and then stained.

30. FIELD STAIN: (THIN FILM)
Materials:
1) Methanol (absolute)
2) Field’s stain A and B
3) Tube with water
4)Staining dishes
5)Filter paper
Procedure:
A. Fix thin film with methanol
for 1 min.

31. B. Dry microscopic slide on filter paper
C. Immerse slide in Field’s stain B (Eosin) for 5 seconds
D. Immediately wash with water
E.Immerse slide in Field‘s stain A (Methylene blue) for 10
secs.
F.Immediately wash with water
G. Dry thin films

32. FIELD STAIN: (THICK FILM)
Materials:
1)Field‘s stain A and B
2) Tube with water
3) Filter paper
Procedure:
A. Immerse thick film in Field‘s stain A (Methylene blue) for 3 secs
Do not forget:
Thick films need to be haemolysed and are therefore not fixed with methanol.

33. B. Rinse immediately in tap water
C. Immerse thick film in Field’s stain B (Eosin) for 3 seconds
D. Then rinse immediately with tap water
E. Let the slide carefully dry

34. JENNER STAIN:
PRINCIPLE: It is similar to wright stain ,used for
staining blood smears.
The Jenner stain Solution is a mixture of several
thiazin dyes in a methanol solvent.
Ionic and nonionic forces are involved in the
binding of these dyes.

35. The staining solution has anionic and cationic
properties.
The negatively charged phosphoric acid groups of
DNA attract the purple polychromatic cationic dyes to
the nuclei.
The blue basophilic granules are stained by the
polychromatic cationic dyes.

36. IMMERSION STAINING PROTOCOL:
1) Thoroughly dry blood or bone marrow smears.
2) Fix smears in absolute methanol for 15 seconds to
5 minutes.
3) Stain smears in Jenners Stain Solution for 2
minutes.

37. 4)Stain in mixture of 50ml of Jenners Stain Solution, 75ml
of pH6.6 Phosphate Buffer Solution and 175ml deionized
water for 5 minutes.
5)Rinse in standing deionized water for 1.5 minutes or
rinse briefly in running deionized water.
6) Air dry smears.
7)Examine smears under a microscope.

38. HORIZONTAL STAINING PROTOCOL:
dried film in a
1)Place slide with thoroughly
horizontal staining rack.
2)Flood smear with absolute methanol for 15-30
seconds and then drain.
3)Flood smear with 1ml Jenners Stain Solution and
let stand for 3 minutes.

39. 4) Add 1mL of pH 6.6 Phosphate Buffer solution and 1
ml deionized water to smear and let stand for 45
seconds.
5) Rinse briefly with running deionized water.
6) Air dry and examine under a microscope.

40. J.S.B. STAIN:
Materials and reagents required:
1)Eosin yellow (water soluble)
2)Methylene blue
3)Potassium dichromate
4)hydrogen phosphate (dihydrate)

41. 5)1% sulphuric Acid.
6)Round bottom flask (2 lit.)
7)Heating mantle
8)Distilled water
9)Staining jars.

42. STAINING TECHNIQUE:
1)Prepare thin and thick smears from malaria cases
on micro slides.
2) De-haemoglobinise the thick smear.
3) Fix the thin smear in methanol for few minutes.
4)Take 3 staining jars for J.S.B. I, J.S.B.II and tap
water.

43. 5)Dip the smears in J.S.B. II for few seconds and
immediately wash in water.
6)Drain the slides free of excess water.
7)Dip the smears in J.S.B.I for 30-40 seconds.
8)Wash well in water and dry.
9)Examine the smears under oil immersion lens of
microscope.

44. STAINING OF THICK SMEAR:
It can be stained with any of the Romanowsky
stains
except that before staining, the smear is
dehaemoglobinised by putting it in distilled water
for 10 minutes.
Autostainers
Currently, automatic staining machines are
available which enable a large batch of slides to
be stained with a uniform quality.

45. Jaswant Singh Battacharya (JSB) Stain for thick and thin films:
• This is the standard method used by the laboratories under the
National Malaria Eradication Programme in India

46. PRECAUTIONS IN STAINING OF PERIPHERAL BLOOD
FILM
1)Dark blue blood film:
It can be due to overstaining, inadequate
washing or improper pH of the buffer. In this RBCs
are blue, nuclear chromatin is black,granules of the
neutrophils are overstained and granules of the
eosinophils are blue or grey.

47. 2)Light pink blood film:
In this RBCs are bright red, the nuclear chromatin is
pale blue and granules of the eosinophils are dark red.
It can be due to understaining, prolonged washing,
mounting the film before drying and improper pH of
the buffer.
3)Precipitate on the blood film:
This could be due to inadequate filtration of the
stain, dust on the slide, drying during staining and
inadequate washing.

48. TOO ACIDIC SUITABLE TOO BASIC

49. REFERENCES:
DACIE AND LEWIS
BANCROFT
Lynch’s Medical laboratory.